Research Marije Meuwissen

Abstract

Malformations of cortical development (MCD) are a heterogenous group of brain malformations, which represent a significant burden for health care and society. Affected individuals suffer from drug-resistant epilepsy and varying degrees of intellectual and motor disability. Using current molecular techniques including SNP-array and whole exome sequencing (WES), over half of MCD cases remain unsolved. Factors contributing to the "unsolved" MCD cases include coding variants of unknown significance (VUS) in known MCD genes, non-coding variants in the known genes influencing splicing or gene expression as well as more complex mutation types (e.g., structural variants, copy number variants), urging the use of whole genome sequencing (WGS) for MCD genetic testing. However, this approach will inevitably lead to the identification of more rare variants of unknown significance (VUS) in known MCD genes. Finally, novel disease genes not previously linked to MCD are still to be discovered. In this project, we aim to establish of a gene-specific disease signature based on RNA-sequencing data that pinpoints the disease gene or pathway on which WGS-based variant analysis should be focused. The selected genes affect either the PI3K-AKT-mTOR pathway or microtubule dynamics, two major pathways involved in brain development. Furthermore, we aim to increase the diagnostic yield in MCD patients by integrating transcriptomics and WGS data of currently "unsolved" MCD cases, allowing the identification of additional variants of interest. Finally, we use the identified disease signatures in the validation of novel MCD candidate genes with similar pathophysiological mechanisms. The results of this project will guide the implementation of transcriptome analysis as another tool in the genetic diagnostic toolbox for MCD and hereby improve patient management and appropriate counseling of families.

Researcher(s)

• Promoter: Jansen Anna
• Co-promoter: Meuwissen Marije
• Fellow: Rosenblum Jessica

Research team(s)

• Translational Neurosciences (TNW)

Project type(s)

• Research Project

Abstract

COL4A1- and COL4A2-related disorders cause a broad spectrum of problems comprising abnormal brain development, brain hemorrhage at any age, aneurysms (local dilatations) of the brain arteries, but also eye or renal problems. In addition, COL4A1 was recently identified as a genetic modifier in Marfan syndrome. We studied the presence of COL4A1 and COL4A2 variants in two patient cohorts; a cerebral palsy (CP) and a TAA (thoracic aortic aneurysm) cohort. This led to a specific interest in the COL4A2 NC1 domain. A burden analysis demonstrated a statistically significant overrepresentation of COL4A2 NC1 variants in the CP cohort. Furthermore, we identified the NC1 variant p.Arg1662His in 3 TAA patients and 3 CP patients of Moroccan descent. In 5 cases in combination with the helical variant p.Met1355Thr. The latter is suggestive of a shared "risk haplotype". The p.Arg1662His variant was significantly overrepresented in Moroccan patients in our cohorts compared to a Moroccan control cohort. In addition, We will study the cellular effects of NC1 variants using patient fibroblasts in order to assess (1) the levels of endoplasmatic reticulum stress and activation of the unfolded protein response and (2) alterations in Akt-FAK-mTOR signaling and procaspase 8 and 9 expression. Fibroblasts were collected from patients harboring (1) the COL4A2 variant p.Arg1662His, (2) the COL4A2 variant p.Arg1662His in combination with the COL4A2 variant p.Met1355Thr and (3) the pathogenic COL4A2 p.Gly1353Ala as a positive control. Three wild-type fibroblasts are used as negative controls. Secondly, we will develop a zebrafish model to study the effect of COL4A2 NC1 variants. We will start with the introduction of the pathogenic COL4A2 p.Gly1353Ala variant and study the effect on zebrafish development using a fish that has fluorescent blood vessels in order to easily pick up abnormal vessels. We will study the occurrence of brain haemorrhage, changes in movement patterns and the basement membrane, a structure that stabilizes the wall of blood vessels and measure the aortic diameter. When a reliable read-out is identified, we will introduce NC1 variants in the zebrafish model to assess their effect. This project is the first study to investigate the contibution of specific COL4A2 NC1-domain variants in pathology. When our findings are corroborated by functional studies, it would also be the first identification of a population-specific COL4A2-related risk haplotype associated with cerebral and aortic vascular pathology, which is an important finding in the age of personalized medicine. Another novelty is the development and use of a zebrafish model to study functional effects of COL4A2 variants using (CRISPR)/Cas9 technology. The model would enable not only functional analysis of additional variants of unknown significance in cerebrovascular pathology and TAA, but additionally allows studies regarding the pathogenic mechanisms underlying different types of COL4A2-mutations. This will help in identifying potential therapeutic strategies. Eventually, the model is suited for testing of potential treatment strategies in vivo, enabling monitoring of the therapeutic effect, as well as unwanted side-effects.

Researcher(s)

• Promoter: Meuwissen Marije
• Co-promoter: Schepers Dorien
• Fellow: Nemegeer Merlijn

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

Cerebral palsy (CP) is a clinical descriptive term that defines a heterogeneous group of non- progressive, neurodevelopmental disorders of motor impairment, which co-occur with a wide range of medical conditions, such as intellectual disability (ID), speech and language deficits, autism, epilepsy and visual and/or hearing impairment. It is the most frequent cause of motor impairment in children, with an important impact on quality of life and a prevalence ranging from 1.5 to 2.5 in 1000 live births. The causes of CP are quite variable. Recent studies demonstrate an important contribution of genetic causes. However, a common mechanism of action of the genes associated with CP is largely unknown. This prompted us to perform genetic analysis in our CP-patient cohort using Single Nucleotide Polymorphism (SNP) array and Whole Exome Sequencing (WES). In this project, we investigate the hypothesis that a subset of genetic causes of CP affect ATG9A transport and subsequently lead to a dysregulation of autophagy. Autophagy is a self-degradative cellular process that removes redundant/dysfunctional proteins, organelles or pathogens. Autophagy was already demonstrated to be an important neuroprotective mechanism against hypoxia-ischemia and glutamate excitotoxicity in animal models. This hypothesis is based on: 1. The important contribution of pathogenic de novo missense variants in KIF1A in our CP cohort (identified in 7/ 141 CP cases). KIF1A is a member of the kinesin motor protein family and is responsible for cargo transport along microtubular tracts. A major cargo of KIF1A is ATG9A, a key regulator of autophagy induction at the presynaptic synapses. 2. In the AP-4 deficiency syndrome, the first known genetic cause of CP, mislocalisation of ATG9A was already demonstrated to be the causal mechanism for the CP phenotype. 3. The identification of likely pathogenic variants in known CP genes (KIF5C) and interesting novel CP candidate genes (KLC3, KLC4, MAP7D2, MAP7D3) that may affect ATG9A transport. In the project, we will study the effect of 1) KIF1A variants and of 2) variants in other CP (candidate) genes on ATG9A and the autophagy process in patients' and controls' fibroblasts. Furthermore, we perform RNA-sequencing in blood and fibroblasts to determine a common expression profile leading to an "autophagy dysregulation" signature. This signature could have important future clinical relevance in screening for potential autophagy dysregulation and monitoring future treatment strategies targeting autophagy.

Researcher(s)

• Promoter: Meuwissen Marije
• Fellow: Thys Liene

Research team(s)

• Medical Genetics (MEDGEN)

Project type(s)

• Research Project

Abstract

COL4A1- and COL4A2-related disorders cause a broad spectrum of problems comprising abnormal brain development, brain hemorrhage at any age, aneurysms (local dilatations) of the brain arteries, but also eye or renal problems. In clinical practice, both genes are studied in disorders of brain vasculature or development and are included in gene panels to study individuals with intellectual disability. These investigations sometimes identify variants of unknown significance (VUS). Because of the important consequences of truly disease-causing mutations, it is of great importance to interpret these variants correctly. In addition, in research setting it was found that COL4A1- and COL4A2-mutations may influence the occurrence of aortic aneurysms. However, further studies are needed. We will develop a zebrafish model to study the effect of variants of unknown significance. No zebrafish model currently exists to study COL4A1- and COL4A2-related disorders. We will start with introducing known disease-causing mutations and study their effect on zebrafish development using a fish that has fluorescent blood vessels in order to easily pick up abnormal vessels. We will study the occurrence of brain haemorrhage, changes in movement patterns and the basement membrane, a structure that stabilizes the wall of blood vessels and measure the aortic diameter. After identifying the abnormalities in true disease-causing mutations, it is possible to study whether VUS contribute to disease.

Researcher(s)

• Promoter: Meuwissen Marije

Research team(s)

Project type(s)

• Research Project